Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates

To acidify the sample, add approximately 20 microliters of 5% trifluoroacetic acid, or TFA, per milliliter of lysate. Mix the tube well and use a pH strip to measure the pH. If necessary, add extra 5% TFA to adjust the pH to 2.5. Next, connect to the shorter end of a C18 column to a vacuum manifold. After setting the vacuum between 17 and 34 kPa, use 3 milliliters of 100% acetonitrile and a glass pipette to wet the column.

Equilibrate the column by using a glass pipette and applying two 3-milliliter aliquots of 0.1% TFA. Then, load the acidified sample onto the column. Use three 3-milliliter volumes of 0.1% TFA to wash the column. Then, apply 2 milliliters of 40% ACN, 0.1% TFA to elute the sample and collect two 2-milliliter fractions into glass culture tubes. With parafilm, cover the eluate tubes, and use a 20 gauge needle to punch three to five holes into the cover before lyophilizing the samples overnight.